Construction of Sox9 gene eukaryotic expression vector and its inductive effects on directed differentiation of bone marrow stromal cells into precartilaginous stem cells in rats.
- Author:
Weihua, HU
;
Fengjing, GUO
;
Feng, LI
;
Hui, HUANG
;
Weikai, ZHANG
;
Anmin, CHEN
- Publication Type:Journal Article
- MeSH:
Base Sequence;
Bone Marrow Cells/*cytology;
Cartilage/*cytology;
Cell Differentiation/genetics;
Cells, Cultured;
Cloning, Molecular;
Genetic Vectors/genetics;
Molecular Sequence Data;
Receptor, Fibroblast Growth Factor, Type 3/metabolism;
Recombinant Proteins/biosynthesis;
Recombinant Proteins/genetics;
SOX9 Transcription Factor/biosynthesis;
SOX9 Transcription Factor/*genetics;
Stem Cells/*cytology;
Stromal Cells/*cytology;
Transfection
- From:
Journal of Huazhong University of Science and Technology (Medical Sciences)
2009;29(3):291-5
- CountryChina
- Language:English
-
Abstract:
Sox9 gene was cloned from immortalized precartilaginous stem cells and its eukaryotic expression vector constructed in order to explore the possibility of bone marrow-derived stromal cells differentiation into precartilaginous stem cells induced by Sox9. A full-length fragment of Sox9 was obtained by RT-PCR, inserted into pGEM-T Easy clone vector, and ligated with pEGFP-IRES2 expression vector by double digestion after sequencing. The compound plasmid was transfected into born marrow-derived stromal cells by Lipofectamine 2000, and the transfection efficacy and the expression of Sox9 and FGFR-3 were observed. Flow cytometry was used to identify the cell phenotype, and MTT was employed to assay proliferative viability of cells. Sequencing, restrictive endonuclease identification and RT-PCR confirmed that the expansion of Sox9 and construction of Sox9 expression vector were successful. After transfection of the recombinant vector into bone marrow-derived stromal cells, the expression of Sox9 and FGFR-3 was detected, and proliferative viability was not different from that of precartilaginous stem cells. It was concluded that Sox9 gene eukaryotic expression vector was successfully constructed, and the transfected bone marrow-derived stromal cells differentiated into the precartilaginous stem cells.
